Many diseases and disorders having an etiology associated with excessive cell proliferation are fatal. The most common of these are cancer and tumors. Noncancerous proliferative diseases can also be life-threatening, however, or lead to a diminished quality of life. These may include, for example: 1) autoimmune diseases such as antigen-induced arthritis and allergic encephalomyelitis, 2) chronic inflammatory proliferative diseases such as rheumatoid arthritis, systemic-onset juvenile chronic arthritis, osteoporosis, and psoriasis, 3) proliferative diseases of the breast including fibrocystic disease, 4) proliferative diseases of the prostate including benign prostatic hyperplasia (BPH), 5) proliferative diseases of the eye including proliferative diabetic retinopathy, and 6) vascular proliferative diseases including atherosclerosis and coronary stenosis. Many efforts have been made to develop curative or ameliorative therapies for these diseases and disorders; however, no comprehensive or universally curative therapy has been developed, even though there are numerous chemotherapeutic approaches that have been proven to be effective against various different cancers, tumors and other types of proliferative disease.
Chemotherapeutic agents are prescribed individually or in combination by clinicians in attempts to develop regimens that are tailored to individual patients' needs. Even so, a key hurdle toward the development of these tailored regimens is the unpredictability of the efficacy of chemotherapeutic agents against specific cancer or tumor phenotypes. Clinicians are forced to deal with these deadly diseases by using hit or miss approaches. They must rely upon a historic review of the recognized or indicated uses of particular chemotherapeutic agents and then speculate or guess as to whether or not a particular single chemotherapeutic agent or combination of chemotherapeutic agents will be therapeutically effective against the cancer or tumor the clinician is attempting to cure. Such a conventional has limited success in the clinic.
Clinicians are in need of a prognostic assay that can predict with some reasonable level of certainty whether or not a particular cancer or tumor phenotype will be therapeutically responsive to a particular single chemotherapeutic agent or combination of therapeutic agents. This type of prognostic assay is extremely useful for chemotherapeutic agents that have a limited use history, such as those that are just entering the clinical environment. It would be extremely beneficial to clinicians to have such a prognostic assay for one or more of such chemotherapeutic agents.
Preclinical studies and retrospective examination of patient data have suggested the potential value of cardiac glycosides, (e.g. bufalin, digoxin, digitoxin, ouabain and oleandrin), in the treatment of various cancers including breast, lung, prostate and leukemia, for example.
One of the pharmacological mechanisms of action of cardiac glycosides involves their ability to bind to the ion exchange pump, Na, K-ATPase and to inhibit the activity of this particular enzyme. Na, K-ATPase, the transmembrane protein that catalyzes the active transport of Na+ and K+ across the plasma membrane, is a well established pharmacologic receptor for cardiac glycosides. This enzyme hydrolyzes ATP and uses the free energy to drive transport of K+ into the cell and Na+ out of cells, against their electrochemical gradients (Hauptman, P. J., Garg, R., and Kelly, R. A. Cardiac glycosides in the next millieum. Prog. Cardiovasc. Dis. 41: 247-254, 1999).
Na, K-ATPase is composed of two heterodimer subunits, the catalytic α-subunit and the glycosylated β-subunit. There is also a γ subunit, but it has not been studied in detail. The α-subunit has binding sites for ATP, Na+, K+, and cardiac glycosides. The β-subunit functions to stabilize the catalytic α-subunit and may play a regulatory role as well. Four different α isoforms (α1, α2, α3, α4) and three different β isoforms (β1, β2, and β3) have been identified in mammalian cells. The relative expression of each type is markedly altered in normal and diseased states. The expression of α isoforms is tissue-type specific and varies among rodent and human tissues (Blanco, G. and Mercer, R. W. Isozymes of the Na, K-ATPase: heterogeneity in structure, diversity in function. Am. J. Physiol. 275 (Renal Physiol. 44): F633-F650, 1998). An altered expression ofNa, K-ATPase isoforms in human cancers such as renal, lung, hepatocellular, and colon has also been reported in contrast to those in corresponding normal tissues (Rajasekaran, S. A., Ball, W. J., Bander, N. H., Pardee, J. D. and Raj asekaran, A. K. Reduced expression of beta subunit of Na/K-APTase in human clear cell renal cell carcinoma. J. Urol. 162: 574-580, 1999; Avila, J., Lecuona, E., Morales, M., Soriano, A., Alonso, T., and Martin-Vasallo, P. Opposite expression pattern of the human Na/K-ATPase beta-1 isoform in stomach and colon adenocarcinomas. Ann. N.Y. Acad. Sci. 834: 633-635, 1997; Espineda, C., Seligson, D. B., Ball, W. J., Rao, J., Palotie, A., Horvath, S., Huang, Y., Shi. T and Rajasekaran, A. K. Analysis of the Na, K-ATPase α- and β-subunit expression profiles of bladder cancer using tissue microarrays. Cancer 97: 1851868, 2003; Jung, M. H., Kim, S. C., Jeon, G. A., Kim, S. H., Kim, Y., Choi, K. S., Park, S. I., Joe, M. K., and Kimm, K. Identification of differentially expressed genes in normal and tumor human gastric tissue. Genomics 69: 281-286, 2000). Additionally, the apparent affinity of cardiac glycosides to the different α isoforms is quite different. Binding of cardiac glycosides to the α1 isoform is less than that which occurs with the α2 and α3 isoforms which are 250-fold or higher more sensitive to inhibition by this type of drug (Blanco, G. and Mercer, R. W. Isozymes of the Na, K-ATPase: heterogeneity in structure, diversity in function. Am. J. Physiol. 275 (Renal Physiol. 44): F633-F650, 1998). Sakai et al. (FEBS Letters 563: 151-154, 2004) report that expression of the α3 subunit isoform is increased in human colorectal cancer cells as compared to normal colorectal cells.
Oleandrin and oleandrigenin inhibit proliferation of human prostate cancer cells through induction of apoptosis which is due, at least in part, to an increase in intracellular Ca2+ via inhibition of Na, K-ATPase (McConkey, D. J., Lin, Y., Nutt, L. K., Ozel, H. Z., and Newman, R. A. Cardiac glycosides stimulate Ca2+ increases and apoptosis in androgen-independent, metastatic human prostate adenocarcinoma cells. Cancer Res. 60: 3807-3812, 2000). Oleandrin and oleandrigenin also inhibit export of fibroblast growth factor-2 through membrane interaction and inhibition of Na, K-ATPase activity (Smith, J. A., Madden, T., Vijjeswarapu, M., and Newman, R. A. Inhibition of export of fibroblast growth factor-2 (EGF-1) from the prostate cancer cell lines PC3 and DU145 by Anvirzel and its cardiac glycoside component, oleandrin. Biochem. Pharmacol. 62: 469-472, 2001).
While Na, K-ATPAase subunit α1 is present in many tissues because the α1β1 complex is considered as ‘house-keeping’ genes, α3 is predominantly detected in excitable tissues, renal cortex, medulla, and papilla as well as nervous tissues
Nerium oleander is an ornamental plant widely distributed in subtropical Asia, the southwestern United States, and the Mediterranean. Its medical and toxicological properties have long been recognized. It has been used, for example, in the treatment of hemorrhoids, ulcers, leprosy, snake bites, and even in the induction of abortion. Oleandrin, an important component of oleander extract, is a potent inhibitor of human tumor cell growth (Afaq F et al. Toxicol. Appl. Pharmacol. 195:361-369, 2004). Oleandrin-mediated cell death is associated with calcium influx, release of cytochrome C from mitochondria, proteolytic processes of caspases 8 and 3, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation.
It has been demonstrated that oleandrin is the principal cytotoxic component of Nerium oleander (Newman, et al., J. Herbal Pharmacotherapy, vol. 13, pp. 1-15, 2001). Oleandrin is a cardiac glycoside that is exogenous and not normally present in the body. Oleandrin induces apoptosis in human but not in murine tumor cell lines (Pathak et al., Anti-Cancer Drugs, vol. 11, pp. 455-463, 2000), inhibits activation of NF-κB (Manna et al., Cancer Res., vol. 60, pp. 3838-3847, 2000), and mediates cell death in part through a calcium-mediated release of cytochrome C (McConkey et al., Cancer Res., vol. 60, pp. 3807-3812, 2000). A Phase I trial of a hot water oleander extract (i.e. Anvirzel™) has been completed recently (Mekhail et al., Am. Soc. Clin. Oncol., vol. 20, p. 82b, 2001). It was concluded that oleander extracts can be safely administered at doses up to 1.2 ml/m2/d. No dose limiting toxicities were found.
Ouabain, a cardiac glycoside endogenous to the body, was reported to enhance in vitro radiosensitivity of A549 human lung adenocarcinoma cells but was ineffective in modifying the radioresponse of normal human lung fibroblasts (Lawrence, Int. J. Radiat. Oncol. Biol. Phys., vol. 15, pp. 953-958, 1988). Ouabain was subsequently shown to radiosensitize human tumor cells of different histology types including squamous cell carcinoma and melanoma (Verheye-Dua et al., Strahlenther. Onkol., vol. 176, pp. 186-191, 2000). The cardiac glycoside oleandrin also has the ability to enhance the sensitivity of cells to the cytotoxic action of ionizing radiation (U.S. patent application Ser. No. 10/957,875 to Newman, et al. and Nasu et al., Cancer Lett. Vol 185, pp. 145-151, 2002). U.S. Pregrant Patent Application Publication No. 20050112059 to Newman et al. discloses the enhancement of radiotherapy in the treatment of cancer by administration of oleandrin.
Chen et al. (Breast Cancer Research and Treatment (2006), 96, 1-15) suggest that cardiac glycosides, such as ouabain and digitalis, might be useful toward developing anti-breast cancer drugs as both Na+, K+-ATPase inhibitors and ER antagonists.
Smith et al. (Biochemical Pharmacology (2000), 62, 1-4) report that ANVIRZEL, and its key cardiac glycoside component oleandrin, inhibits the exportation of a tumor growth factor, fibroblast growth factor-2 (FGF-2), from the prostate cancer cell lines PC3 and DU145.
Newman et al. (J. Experimental Therapeutics and Oncology (2006), 5, 167-181) report that incubation of human malignant melanoma BRO cells with oleandrin results in a time-dependent formation of reactive oxygen species, superoxide anion radicals, that mediate mitochondrial injury, loss of cellular glutathione (GSH) pools and, ultimately, tumor cell death.
Extraction of glycosides from plants of Nerium species has provided pharmacologically/therapeutically active ingredients from Nerium oleander. Among these are oleandrin, nerine, and other cardiac glycoside compounds. The plant extracts are useful in the treatment of cell-proliferative diseases in animals. Oleandrin extracts obtained by hot-water extraction of Nerium oleander, sold under the trademark ANVIRZEL™, are commercially available and contain the concentrated form or powdered form of a hot-water extract of Nerium oleander. 
Huachansu is an extract obtained from toad skin and it comprises bufadienolides, such as bufalin, a cardiac glycoside. HuaChanSu is an approved medication for the treatment of cancer in China. It has been used to treat various cancers, including hepatic, gastric, lung, skin, and esophageal cancers.
In view of the important utility of cardiac glycosides in treating diseases or disorders having an etiology associated with cell proliferation, a need remains for a method of predicting the therapeutic response of the disease or disorder to a cardiac glycoside. No such method is disclosed in or suggested by the prior art.